This invention is directed to a modified Factor VIII cDNA and its use for the improvement of the Factor VIII production.
Factor VIII (FVIII) is a X-linked gene product implicated in the blood coagulation cascade. The factor VIII is synthetized as a 2351 amino acid single-chain polypeptide having the domain structure A1-A2-B-A3-C1-C2 and comprising a 19 amino acid signal peptide (Gitschier J. et al., 1984; Toole, J. J. et al., 1984; Vehar, G. A. et al., 1984). Plasma FVIII is a heterodimer consisting of a carboxy-terminal derived light chain of 80 kDa in a metal-ion dependent association with a variable sized amino-terminal heavy chain (200-90 kDa; Andersson et al., 1986). Absence or deficiency of FVIII causes severe bleeding disorders called hemophilia A.
The level of FVIII production remains low after cell transfection compared to other genes. Three reasons have been identified so far: (1) FVIII mRNA is inefficiently produced, (2) FVIII translocation from endoplasmic reticulum to Golgi apparatus is low and (3) FVIII is sensitive to proteolysis. Therefore, the improvement of FVIII transgenes is an important challenge for hemophilia A gene therapy.
It has already been proposed in the European patent application 99 104 050.2 to modify the FVIII cDNA by deleting the B-domain of the wild type cDNA and by inserting a truncated FIX intron 1 in different locations of the Factor VIII cDNA. Such modified Factor VIII cDNA may be used for a higher yield production of FVIII in vitro as well as in a transformation vector for gene therapy. A cDNA bearing the FIX truncated intron 1 in both the intron 1 and intron 13 locations led to the highest FVIII production after transfection of CHO and HepG2 cell lines. This recombinant FVIII was a biologically active protein.
In order to further improve the yield in the gentechnological production of Factor VIII the present invention is directed to the expression of Factor VIII in hematopoietic cells and especially in platelets using the tissue-specific promoter of the glycoprotein II b (GPIIb).
The feasitility of this approach was demonstrated, first by use of the hematopoietic cell line HEL. The human erythroleukemia cell line is known to express an erythroid phenotype (Martin et al., 1982) but also some megakaryocytic markers such as platelet membrane glycoproteins (Tabilio et al., 1984). Upon induction by phorbol-12-myristate-13-acetate (PMA), HEL cell line expresses increased amounts of megakaryocytic proteins like glycoproteins IIb/IIIa, Platelet factor 4, or von Willebrand factor (vWF) (Long and coll., 1990).
Another hematopoietic cell line, Dami, established from the blood of a patient with a megakaryoblastic leukaemia, appears to be a pure population of megakaryocyte-like cells (Greenberg et al., 1988). Cultured Dami cells express platelet glycoproteins GPIIb and GPIIb/IIIa. After PMA stimulation, surface expression of these two platelet glycoproteins, and vWF synthesis were increased. These changes were associated with a decrease in the proliferation of the stimulated Dami cells (Greenberg et al., 1988; Ballen et al., 1996; von der Vuurst et al., 1998). The multimerin molecule, which colocalizes with vWF in platelet xcex1-granules, was shown to be synthetized in PMA-stimulated Dami cells where it presented a granular distribution (Hayward et al., 1993). The same results were obtained with the plasminogen activator inhibitor type I and vWF (Hill et al., 1996). Dami cells were used to study the megakaryocyte-specific expression of FVIII under the GPIIb promoter control.
The present invention discloses the ability of hematopoietic cell lines to produce an active FVIII molecule. It could be demonstrated that Dami cells transfected with the GPIIb constructs are able to synthetize FVIII and that FIX intron 1 sequences increase dramatically the production of Factor VIII.
A modified Factor VIII cDNA has been found wherein the B-domain of the wild type factor cDNA has been deleted and a truncated Factor IX intron has been inserted in two locations of the Factor VIII cDNA containing as a promoter a cDNA which is suitable for the expression in hematopoietic cell lines and specifically in platelets. The cDNA coding for the human platelet glycoprotein IIb (GPIIb) is preferred as a promoter. The modified Factor VIII cDNA of the present invention contains the truncated Factor IX intron 1 in the Factor VIII introns 1 and 13.
A further object of the invention is a process for the production of Factor VIII in the cell lines HEL or Dami using the above-mentioned modified Factor VIII cDNA. Preferred is a process wherein the production of Factor VIII is stimulated by an inducer. The best results have been obtained when phorbol-12-myristate-13-acetate (PMA) was used.
Materials and Methods
Vectors: The pcDNA3-FVIII and pcDNA3-FVIII I1+13 were the same vectors as disclosed in the European patent application 99 104 050.2. The pBLCAT3-vector bearing the xe2x88x92643/+33 GPIIb promoter was obtained from G. Uzan (Uzan et al., 1991). This promoter was sorted from the pBLCAT3-GPIIb vector after HindIII-BamHI digestion (Promega, Charbonnixc3xa8res, France) and was introduced in pcDNA3.1 vector (Invitrogen, Groningen, The Netherlands) opened by the same enzymes. This construct was then deleted of the CMV promoter by MluIClaI digestion, and the construct obtained was so called pcDNA3-GPIIb. The pTracer(trademark)-EF C vector was obtained from Invitrogen (Groningen, The Netherlands). This vector is bearing a Zeozine(trademark) resistance gene.
Cell Culture: HEL92.1.7 was obtained from ECACC (Sophia Antipolis, France). The cells were maintained in RPMI/10% FCS medium with 5% CO2. For stable transfections with pcDNA3 constructs, HEL cells (1xc3x97106 cells) were transfected with 2 xcexcg of PvuI linearized plasmid using 6 xcexcl FUGENE(trademark) 6 (Roche Diagnostics, Meylan, France) during 5 hours. After incubation, the cells were harvested and placed in fresh medium supplemented with 0,6 mg/ml geneticin (Gibco BRL, Cergy Pontoise, France).
Dami cells were maintained in RPMI/10% FCS medium with 5% C02. For stable transfections with pTracer constructs, Dami cell (1xc3x97106 cells) were transfected with 2 xcexcg of PvuI linearized plasmid using 6 xcexcl FUGENE(trademark) during 5 hours. The cells were then harvested and placed in fresh medium. Zeocin(trademark) (Invitrogen, Groningen, The Netherlands) was subsequently added at a final concentration of 300 xcexcg/ml.
Cell Inductions: To compare FVIII production, the resistant cells (2.5xc3x97105 cells/ml) were placed in RPMI/1% BSA with human vWF xc2x1PMA 1 nM. After 4 days of incubation, the cells were numbered and the supernatants were harvested. The supernatants were concentrated on Microsep(trademark) microconcentrators (Pall Gelman Sciences, France) with a 30 Kd cut-off. The cells were lysated in Hepes 20 mM, KCl 0.1 M, MgCl2 2 mM, Tritonxc3x97100 0.5%. Protein concentrations were measured using Bio-Rad Dc Protein Assay (Bio-Rad, Ivry sur Seine, France). FVIII productions were measured using FVIII ELISA kit (Asserachrom FVIII, Stago Asnixc3xa8res, France). Concentrated culture media were tested for coagulation activity using a chromogenic FVIII assay (Coamatic FVIII, Biogenic, France).
RT-PCR and PCR: Reverse transcriptase (RT) reactions were realized with 2 xcexcg mRNA (extracted with Rneasy Mini kit; QIAGEN S. A., France) using the Superscript(trademark) Rnase H Reverse transcriptase (Gibco BRL, Cergy Pontoise, France) and oligo(dT)15 primer (Promega, Charbonnixc3xa8res, France). For PCR, Expand(trademark) long template PCR system (Roche Diagnostics, Meylan, France) was used with 4 xcexcl of each RT product or 10 ng of each control plasmid. Intron splicing was studied using a set of primers specific for intron 1 location and another set for intron 13 location. The first PCR gives a 1701 bp fragment without the intronic sequence and a 2014 bp fragment with the FIX intron 1 sequence. With the 2 other primers, the size of PCR fragments was 623 bp and 935 bp depending upon the absence or the presence of intron 13, respectively. RT-PCR and PCR fragments were run on 0.8% agarose gel and were compared with the 1 Kb ladder (Gibco BRL, Cergy Pontoise, France).
Immunoprecipitations and FVIII Immunoblot Analysis: Before immunoprecipitations, lysates were incubated with human vWF (2,000 ng vWF for 40 ng FVIII; Diagnostica Stago, Asnixc3xa8res, France) for 10 min at room temperature. Fifty microliters of anti human vWF antibody beads provided by Aventis Behring (USA) were added to the samples and incubated overnight at 4xc2x0 C. The beads were then collected after centrifugation (2 min at 2500 rpm), washed three times with the equilibration buffer (Hepes 10 mM, KCl 100 mM, MgCl2 2 mM, Tritionxc3x97100 0.1%) and diluted in Laemmli buffer (Laemmli, 1970). Samples were then subjected to electrophoresis on SDS-PAGE/7% polyacrylamide gel and semi-dry blotted onto Hybond(trademark) C Pure membrane (Amersham Pharmacia Biotech Europe GmbH, France) The immunoblots were blocked with TBS-T (Tris-HCl 10 mM pH 7.5, NaCl 0.15 M, Tween 0.1%) for 1 h at room temperature and then incubated with 1:3,000 dilution of a sheep anti-human FVIII antibody (Cedarlane, Ontario, Canada). The membrane was then washed 3 times in TBS-T and incubated with a 1:10.000 dilution of a peroxydase-labeled anti-sheep antibody (Dako S. A., Trappes, France) for 30 min. After 3 washes, chemiluminescent signal was detected by autoradiography using the ECL System (Amersham Pharmacia Biotech Europe GmbH, France).